DNA methyltransferases (also referred to as DNA methylases) transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on a DNA molecule. Several biological functions have been attributed to the methylated bases in DNA. The most established biological function is the protection of the DNA from digestion by cognate restriction enzymes. The restriction modification phenomenon has been observed only in bacteria. Mammalian cells possess at least several methyltransferases; one of these (DNMT1) preferentially methylates cytosine residues on the DNA, which are 5′ (upstream) neighbors of guanine (forming the dinucleotide CpG). This methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin and Riggs, eds. in DNA Methylation Biochemistry and Biological Significance, Springer-Verlag, New York, 1984).
When most gene sequences contain many methylated cytosines, they are less likely to be expressed (Willson, Trends Genet. 7:107-109, 1991); in particular, if a site in the promoter of the gene is methylated, gene silencing is likely to occur. Hence, if a maternally-inherited copy of a gene is more highly methylated than the paternally-inherited copy, the paternally-inherited copy will be expressed more effectively. Similarly, when a gene is expressed in a tissue-specific manner, that gene often will be unmethylated in the tissues where it is active but highly methylated in the tissues where it is inactive.
Incorrect methylation is believed to be the cause of some diseases such as Beckwith-Wiedemann syndrome and Prader-Willi syndrome (Henry et al., Nature 351:665, 1991; Nicholls et al., Nature 342:281, 1989), as well as a contributing factor in many cancers (Laird and Jaenisch, Hum. Mo. Genet. 3 Spec. No.: 1487-1495, 1994). Expression of a tumor suppressor gene can be abolished by de novo DNA methylation of a normally unmethylated 5′ CpG island (Issa et al., Nature Genet., 7:536, 1994; Herman et al., Proc. Natl. Acad. Sci., U.S.A., 91:9700, 1994; Merlo et al., Nature Med., 1:686, 1995; Herman et al., Cancer Res., 56:722, 1996; Graff et al., Cancer Res., 55:5195, 1995; Herman et al., Cancer Res., 55:4525, 1995). Such hypermethylation has now been associated with the loss of expression of VHL, a renal cancer tumor suppressor gene on 3p (Herman et al., Proc. Natl. Acad. Sci. USA, 91:9700-9704, 1994), the estrogen receptor gene on 6q (Ottaviano et al., Cancer Res., 54:2552, 1994) and the H19 gene on 11p (Steenman et al., Nature Genetics, 7:433, 1994). Similarly, a CpG island has been identified at 17p 13.3, which is aberrantly hypermethylated in multiple common types of human cancers (Makos et al., Proc. Natl. Acad. Sci. USA, 89:1929, 1992; Makos et al., Cancer Res., 53:2715, 1993; Makos et al., Cancer Res. 53:2719, 1993). This hypermethylation coincides with the timing and frequency of 17p losses and p53 mutations in brain, colon, and renal cancers. Many effects of methylation are discussed in detail for instance in published International patent application PCT/US00/02530.
Both 5-fluorodeoxycytidine (FdCyd) and 5-azacytidine (5-aza-CR) have been shown to inhibit methylation of DNA with resultant effects on gene expression and cell differentiation (Jones and Taylor, Cell 20:85-93, 1980; Osterman et al., Biochemistry 27:5204-5210, 1988). However, these compounds are unstable or produce toxic metabolites in vivo (Santi et al., Proc. Natl. Acad. Sci. USA 91:6993-6997, 1984; Newman et al., Proc. Natl. Acad. Sci. USA 79:6419-6423, 1982). Thus, there exists a need for an effective, stable, and low-toxicity inhibitor of DNA methylation.